Here, membrane perturbation of live E. coli treated with the parent KYR and C14-KYR peptides was compared at the nanoscale level using AFM imaging. AFM analyses, including average cellular roughness and force spectroscopy, revealed the severe surface disruption mechanism of C14-KYR. A loss of surface roughness and changes in topographic features included membrane shrinkage, periplasmic membrane separation from the cell wall, and cytosolic leakage. Additional evidence from synchrotron radiation FTIR microspectroscopy (SR-FTIR) revealed a marked structural change in the membrane component after lipopeptide attack. The average roughness of the E. coli cell before and after treatment with C14-KYR was 129.2 ± 51.4 and 223.5 ± 14.1 nm, respectively. The average rupture force of the cell treated with C14-KYR was 0.16 nN, four times higher than that of the untreated cell. Our study demonstrates that the mechanistic effect of the lipopeptide against bacterial cells can be quantified through surface imaging and adhesion force using AFM.Lipopeptides have been extensively studied as potential antimicrobial agents. In this study, we focused on the C Natural lipopeptides (LiPs) are molecules consisting of a peptide linked to a lipid moiety commonly produced non-ribosomally in bacteria and fungi during cultivation in the presence of carbon sources. LiPs are considered among promising candidates for their potential use as antimicrobial agents and for vaccine therapy applications ,2. CompaThe mechanisms of action of LiPs are similar to those of antimicrobial peptides (AMPs) where the bacterial membrane is a major target of LiPs. The general mechanism of AMPs involves the binding and permeabilization of the bacterial cell membranes. Most peptides are cationic and thus bind to anionic lipids in the target membranes via electrostatic interaction. Additionally, hydrophobic interactions contribute to the peptides’ affinity for the membranes where penetration through the lipid’s head groups unevenly distends and disrupts the membrane . LiPs reAtomic force microscopy (AFM) has recently become a powerful technique for structural and morphological studies in microorganisms ,12. HighPseudomonas aeruginosa as a model of a multidrug-resistant clinical strain for studying the differential effect at the nanoscale by two reference antibiotics. The organization at the nanoscale of the bacterial cell wall was examined using functionalized AFM tips to address changes in cell morphology, surface roughness, and elasticity after antibiotic treatment. The high resolution and sensitivity of AFM have brought novelties into the real-time effects of antibiotics and external agents in the cell envelope ultrastructure [E. coli cells at high resolution in real time at the nanometer level [Streptococcus agalactiae. The AFM imaging and molecular mapping was represented as a platform for analyzing the molecular arrangement of the cell wall of the bacterial pathogens [In recent years, progress has been made in understanding the surface properties of microorganisms using AFM. For example, Yang et al. studied the effect of silver ions on the bacterial membrane and successfully provided new insight into the antimicrobial mechanism of silver ions and the understanding of the relationship between the structure and function of the bacterial cell . Formosatructure . Fantnerer level . The samer level ,22. Receathogens .14) acid at the N-terminus. The resulting LiP, C14-KYR, exhibited a higher antibacterial property than the parent peptide, KYR [14-KYR against E. coli HB101 and for the morphological changes of the cell surface using AFM. The mechanistic comparison between the parent peptide, KYR, and LiP, C14-KYR, was elucidated. AFM images of E. coli HB101 showed that the C14-KYR induced dramatic changes on the cell surface, including shrinkage of the cell surface, separation of the cell wall and cytoplasm, and leakage of intracellular materials. In contrast, cells treated with the KYR revealed a slightly damaged surface, giving a similar pattern to the untreated cell. Additional evidence from SR-FTIR revealed marked structural changes in the membrane component after LiP attack. We clearly demonstrated in high-resolution images that the C14-KYR caused a higher degree of surface damage than its parent peptide.We previously modified KYR, a cationic tripeptide with myristic  NaCl or phosphate-buffered saline (PBS), pH 7.4, depending on the experiments. Gram-negative E. coli HB101 used in antimicrobial studies was a gift from Camesano Laboratory, Worcester Polytechnic Institutes, Massachusetts, USA. All solutions were prepared with ultrapure Millipore water . The Luria-Bertani (LB) medium was purchased  and sterilized under high-pressure steam at 121 °C for 30 min.Peptides were synthesized via Fmoc solid-phase peptide chemistry on rink amide-4-methylbenzhydrylamine hydrochloride salt resin by GL Biochem (Shanghai) Ltd. . The following peptides, the KYR and the CE. coli HB101 cell suspension (1 × 106 CFU/mL) was centrifuged for 5 min. The cell pellets were washed with 0.9% (w/v) NaCl twice and re-suspended in 50 mL of 0.9% (w/v) NaCl. The cell was treated with 20 µM of C14-KYR (MIC) and 100 µM of C14-KYR (5x MIC) for 15 min at room temperature. Then, cells were washed three times with 0.1% (w/v) NaCl and resuspended in 0.1% (w/v) NaCl. The cell suspension was transferred onto Low-e Microscope Slides  and was vacuum-dried for 30 min in a desiccator [The siccator ,24,25,26−1. Samples were analyzed in transflection mode, using the conventional internal IR source of a Bruker Vertex 70 spectrometer connected to the Bruker Hyperion 2000 microscope . IR signals were acquired from a 65 µm × 65 µm region, which covered more than 100 cells in the most homogeneous zone. The measurements were performed with a spectral resolution of 4 cm−1 with 64 scans co-added.The SR-FTIR experiments were conducted at the IR-end station of Synchrotron Light Research Institute (Public Organization) . The infrared spectra were collected using FTIR microspectroscopy coupled with an MCT detector cooled with liquid nitrogen over the measurement range from 4000 to 600 cm14-KYR (MIC-treated cells), and 113 spectra of the condition treated with 5x MIC of C14-KYR (5xMIC-treated cells). The spectra were processed using the second derivative and were vector-normalized by the Savitzky–Golay method , and they were then normalized using extended multiplicative signal correction (EMSC) into one spectrum of each condition in the spectral regions from 1750 to 850 cm−1 [−1), amide I and amide II vibration of the protein region (1700–1500 cm−1), and functional groups (1300–900 cm−1) in nucleic acids (DNA and RNA), carbohydrates, phospholipids, proteins, and phosphorylated proteins.OPUS 6.5 software (Bruker) was employed to control the instrument, sample stage movements, spectral acquisition, and the post-acquisition processing of raw spectra. Integration was applied to the following intervals. Spectra from each group were analyzed using principal component analysis (PCA) to distinguish different chemical components of the samples using the Unscrambler 9.7 software . In our experiment, the measurement produced 100 spectra of the untreated condition , 137 spectra of the condition treated with a MIC of C850 cm−1 ,27. AssiE. coli HB101 cell was prepared for imaging in each treatment condition as previously described with minor modification [w/v) NaCl. 10 mL of bacterial cell suspension (1 × 109 cells/mL) was mixed with 300 µL of 100 mM of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), pH 5.5 (Pierce), and it was then agitated for 10 min. After treatment with EDC, 300 µL of 40 mM of N-hydroxysulfosuccinimide (Sulfo-NHS), pH 7.5 (Pierce), was added to the bacterial suspension and mixed for an additional 10 min [9 cells/mL) were first exposed to either KYR or C14-KYR at 20 μM. The bacterial solution  was added to each glass slide and agitated at 70 rpm for 1–2 h to promote bacterial lawn formation. To visualize the morphology of treated cells, 15–20 μL of either KYR or C14-KYR at 20 μM in PBS was added to the bacterial lawn. After incubation at room temperature, the cells were imaged by AFM.The fication . Brieflyl 10 min . Prior tE. coli cell was imaged in solution using contact mode before and after treatment with KYR or C14-KYR. The microscope was equipped with a piezoscanner with a maximum scan range of 5 μm × 5 μm. Silicon nitride cantilevers  with a spring constant of ~0.12–0.16 N/m and a resonant frequency of ~5 kHz were used in imaging. The scanning speed was set to 0.5–1.0 Hz. Height, deflection, and phase images were obtained simultaneously in all experiments. Quantitative information from the AFM images were acquired via AFM analysis using the imaging software (Gwyddion). On average, 50 single force curves were acquired on top of the bacterial cell from at least 8 cells from two independent cultures for each condition. During force curve acquisition, the tip was made to gently touch the bacterial surface with a force of less than 5 nN to avoid breakage or deformation of the bacteria membrane. All retraction force curves were recorded at an interaction time of 1 s. The data analysis was performed using custom software written for MATLAB  by using a procedure we previously described [Imaging was performed with an MFP-3D Bio AFM . The escribed ,30.14-KYR lipopeptide, a KYR peptide with amidation at the C-terminus and acylation with myristic acid at the N-terminus. Among the acylation of KYR with a 10- to 16-carbon chain length, C14-KYR exhibits antimicrobial activity toward both Gram-positive and Gram-negative bacteria with the highest selectivity index [E. coli HB101 was chosen as a bacterial model for studying changes in the bacterial surface after treatment with LiP. The characteristic change in surface cells after treatment with the antimicrobial agent was observed using an atomic force microscope. The insight event  of the membrane disruption action on cells treated with the antimicrobial lipopeptide was obtained using imaging, characterizing the alteration in the biochemical properties involved with the antimicrobial lipopeptide.In order to provide visual and quantitative evidence for the mechanistic actions of the LiP, including pore formation on the bacterial cell surface and cell morphology at the nanoscale, the AFM technique was employed. In this study, we focused on the Cty index . The E. →4) glycosidic bonds. The cross-bridge is most commonly composed of tetrapeptides such as L-alanine, D-glutamic acid, and meso-diaminopimelic acid (DPA). Gram-negative bacteria comprise the outer membrane (OM) of the peptidoglycan (PG), while the outside layer contains phospholipids, and the inner layer contains lipopolysaccharides (LPS). The outer layer consists of three basic regions: the O-specific side chain, the inner and outer core oligosaccharides, and a lipid anchor called lipid A [In order to understand the FTIR spectra for cell surface characteristics, the cell composition of bacteria is a prerequisite. Generally, Gram-negative bacteria have a thin peptidoglycan. The primary structure comprises parallel polysaccharide chains of N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM) that are cross-linked by β , (B) protein (1700–1500 cm−1), and (C) carbohydrate and nucleic acid (1300–900 cm−1) [In our experiment, we attempted to discriminate the membrane component between untreated cells and C00 cm−1) ,25,26,36E. coli profile and E. coli treated with C14-KYR. The obvious changes were related to shifting due to the deformation of C-H stretching. The treated E. coli at MIC C14-KYR was detected through modifications of bands at 2850, 2921, and 2960 cm−1, which were caused by C-H stretching from a hydrocarbon chain, i.e., fatty acid and lipid region. The main alteration in these regions of the MIC condition lightly shifted the peak, while 5xMIC C14-KYR addition was detectable with the decrease in the shift of the absorption peak, indicating the change in C-H stretching of the fatty-tail structure. The modification of the C-H vibration on the exposed bacterial profile is interpreted as bacterial lysis [al lysis ,34,36.−1, which was dominated by the amide I and amide II of proteins and peptides [−1  membrane permeability and also completely depolarized the bacterial membrane, indicating cell death within 10 min. Membrane depolarization was stably saturated after 15 min of treatment by lipopeptide. In contrast, the KYR peptide can bind to the outer bacterial membrane but cannot traverse across the membrane [E. coli HB101 population in solution  than the untreated cell. We noted that the increase in surface roughness correlated with the C14-KYR-induced change in morphology of the bacterial membrane, which eventually led to cell death. To obtain further quantitative analyses on the cell surface roughness before and after the exposure to KYR and C14-KYR peptides on the single E. coli HB101 cell were visualized in three dimensions, as shown in topography images and height profiles  by using a procedure we previously described [14-KYR at MIC for 30 min  case, substantial effort is needed. In contrast, due to C14 KYR bound to the outer bacterial membrane with electrostatic bonding between the positively charged KYR and the negatively charged bacterial membrane, we were able to permeate the inner membrane. This mechanism inducing a change in the bacterial morphology following treatment indicates membrane imbalances with the internal contents leaking out, accompanied by cell swelling, causing possible water uptake through cell lysis, which provides measurement of the lower rupture force.Quantitative rupture force measurement with AFM is a promising way to characterize how the lipopeptide affects the mechanical properties of the bacterial surface. The AFM probe as nanoindentation depth plots was used and analyzed according to theoretical models for providing quantitative information on the rupture force of the sample . These mescribed ,30. AFM-escribed . Interacescribed . When thescribed . Then, ar 30 min , the shaR at MIC B, the evE. coli HB101 cell surface was visualized after treatment with the C14-KYR lipopeptide. E. coli belongs to the Gram-negative bacteria consisting of a lipopolysaccharide outer membrane, thin peptidoglycan layer (~2 nm), and cytoplasmic membrane [E. coli acts as a selective permeability barrier. As a result, when treated with 20 μM of KYR peptide, the peptide was unable to reach the peptidoglycan layer. Consequently, the E. coli HB101 remained in their rod-shaped structure, although some surface area was disrupted. In the other case, the concentration of 20 μM of C14-KYR lipopeptide could reach the peptidoglycan layer and into the inner membrane. The bacterial inner membrane shrank and became separated from the cell wall, resulting in the leakage of intracellular materials. The corresponding SR-FTIR results revealed structural changes in the membrane component after LiP attack. The most differences were especially found in the protein and peptide regions. This occurrence indicates that C14-KYR destabilizes the cell membrane by altering the structural conformation of membrane components resulting in membrane disruption and leakage. To further provide supporting evidence for the mechanistic action, the elasticity of the membrane surface was determined. We found that the static roughness of the E. coli surface after treatment with the C14-KYR became greater than that of the untreated cell. Additionally, the adhesion force between the biomolecule substance on the cell membrane and the AFM tip was determined from the rupture force analysis [E. coli cell was 0.04 nN, but with the treatment of C14-KYR, the rupture force increased to 0.16 nN. This finding indicated that the C14-KYR disrupted the bacterial membrane and induced changes in the elasticity of the cell surface, resulting in an alteration of their rod-shaped morphology and rigidity, which eventually led to cell death.In this study, intense damage of the membrane ,41. The analysis ,38. The 14-KYR, exhibits higher antibacterial properties, particularly membrane-disrupting activity, than the parent peptide, KYR [14-KYR against E. coli HB101 and for the morphological changes in the cell surface using AFM imaging and IR spectroscopy. Measurements of the cell roughness and force spectroscopy were performed before and after exposure to the C14-KYR lipopeptide at different time courses. AFM creep deformation experiments of C14-KYR induced dramatic changes on the cell surface, including shrinkage of the cell surface, separation of the cell wall and cytoplasm, and leakage of intracellular materials. In contrast, cell treated with the KYR revealed a slightly damaged surface, giving a similar pattern to the untreated cell. Further evidence from SR-FTIR revealed structural changes in the membrane component after LiP attack. The most differences were especially found in the protein and peptide regions when exposed by 5x C14-KYR. This occurrence indicates that C14-KYR destabilizes the cell membrane by altering the structural conformation of membrane components, resulting in membrane disruption and leakage. This evidence provides detailed information on the morphological changes of the cell at the nanoscale, which corresponds to biochemical changes in the bacterial membrane.In our previous study, we highlighted and followed the progression of events that occur during the membrane disintegration process over time. We previously found that LiP, Cide, KYR . Here, w


To report a rare case of primary orbital pleomorphic liposarcoma and present a relevant literature review.An 11-year-old boy presented with an enlarging, painless lower right eyelid mass that was noted 4 months ago. Imaging revealed a 3.2 × 2.1 × 3.7-cm-sized well-circumscribed lobulated mass. Biopsy revealed a pleomorphic spindle cell neoplasm that was consistent with a pleomorphic liposarcoma. A systemic evaluation found no evidence of distant metastasis. Despite four chemotherapy cycles, the mass size increased at follow-up. Total orbital exenteration was performed 1 year after confirming a diagnosis of pleomorphic liposarcoma. At the 6-month follow-up, the patient was alive with no signs of recurrence.Pediatric pleomorphic liposarcoma is a very rare entity. Its definite diagnosis relies on histopathological results. In the absence of systemic metastasis, total orbital exenteration is the optimal approach for local control. Only seven case reports of primary orbital pleomorphic liposarcoma have been found in the literature since 1990, and only two of them were in patients aged <20 years.2An 11-year-old Asian boy with an enlarging lower right eyelid mass was referred to our hospital for further evaluation and complete tumor removal. The child had homozygous hemoglobin E disease, with no anemic symptoms, as an underlying disease. He denied any significant past ocular history or family history of malignancy at a young age. Four months ago, he first noticed this painless mass of approximately 1 cm. Two months after the mass had developed, the child underwent contrast-enhanced orbital magnetic resonance imaging (MRI), which revealed a 3.2× 2.1 × 3.7-cm-sized well-circumscribed lobulated mass on the lower right eyelid, abutting on the right inferior rectus muscle and causing superior right eye globe displacement.On physical examination, his best-corrected visual acuity was 2/60 OD and 6/12 OS, with normal pupil reaction. External examination showed a 5-cm- painless, firm lower right eyelid mass. The mass caused superior globe displacement, complete ptosis, marked conjunctival chemosis, and limited extraocular muscle movement, with the greatest restriction on the inferior directions A. The leNeoadjuvant chemotherapy , including ifosfamide and doxorubicin, was administered as four cycles in total (3 weeks apart). The mass slightly increased in size at the follow-up examination, and a trace relative afferent pupillary defect of the right eye was noted B. Orbita3Liposarcoma of the head and neck region is a rare entity. The pleomorphic subtype is a high-grade undifferentiated sarcoma with a variable number of pleomorphic lipoblasts and has the most aggressive course, with a 1-year survival rate of approximately 45%.The tumor was surgically resected in all of the cases, either via local resection or exenteration. Two patients died, whereas the others showed no sign of recurrence or were lost to follow-up.12Liposarcoma is extremely uncommon in children. The most common primary site in adults is the extremities. Madge et al. reported on a 19-year-old Caucasian man with primary orbital pleomorphic liposarcoma.The treatment option for liposarcomas is primarily surgical resection, and it is generally recommended to excise at least 2 cm of negative surgical margins in cases of soft tissue sarcomas.4We described the clinical features of an 11-year-old boy with primary orbital pleomorphic liposarcoma, a rare entity. Diagnosis primarily depended on histopathological findings obtained via biopsy. In the absence of systemic metastasis, total exenteration of the orbit with adequate surgical margin seemed to be the best treatment option. Adjunctive chemotherapy or radiotherapy might improve local control, although the decision should be weighed against the risk. Clinicians should consider liposarcoma as a differential diagnosis of orbital masses in children.Trakanta Wannapanich: Conceptualization, Methodology, Validation, Writing - Original Draft, Writing - Review & Editing, Visualization.Paitoon Pratipanawat: Validation, Writing - Review & Editing, Supervision, Project administration, Funding acquisition.Both patient and patient's legal guardian consented to publication of the case in writing. The patient and his parents have given their permission to publish their clinical data and images.Faculty of Medicine, Khon Kaen University, Thailand.The authors declare that there are no conflict of interests of this paper.


P=0.007). The most frequent grade 3-4 adverse event in the chemotherapy and chemoimmunotherapy groups was myelosuppression . The most frequent grade 3-4 adverse events in the immunotherapy group were diarrhea  and hepatotoxicity . Chemoimmunotherapy had the highest 2-year PFS as a first-line treatment for advanced pLELC compared to chemotherapy and immunotherapy. This study suggests that chemoimmunotherapy may be the best first-line treatment for patients with advanced pLELC.Pulmonary lymphoepithelioma-like carcinoma (pLELC) is a rare subtype of lung cancer that is associated with the Epstein-Barr virus in Asia. Due to the lack of prospective studies, the best first-line treatment and survival outcomes remain unclear. Herein, This study investigated the efficacy and safety of different treatment regimens for advanced pLELC. This retrospective study included 68 patients with advanced pLELC from two centers in China. Patients were divided into three groups according to different first-line treatments: chemotherapy , immunotherapy , and chemoimmunotherapy . The primary endpoint of this study was the 2-year progression-free survival (PFS) of each group. The results show that the median PFS was 6.9 months  in the chemotherapy group, 11.0 months  in the immunotherapy group, and 11.8 months  in the chemoimmunotherapy group. There was a significant difference in 2-year PFS between the chemoimmunotherapy group and the chemotherapy group (hazard ratio, 0.38, 95% confidence interval: 0.18-0.78, log-rank  Pulmonary lymphoepithelioma-like carcinoma (pLELC) is a rare pathological subtype of lung cancer , which hLung tumor cells frequently express high levels of programmed death ligand 1 (PD-L1), which is the ligand of the programmed death-1 (PD-1) receptor on T cells, allowing tumors to directly inhibit the host immune response by inhibiting the proliferation and function of T cells –9. MoreoNasopharyngeal carcinoma (NPC) is also associated with EBV infection and has a high propensity for regional and distant metastases, while it is very sensitive to radiation and chemotherapy . A commoCurrently, there is no standard treatment for pLELC. Most patients diagnosed with pLELC are usually present at an early stage and undergo complete resection . Howevervia biopsy and computed tomography/positron emission tomography at The Third Affiliated Hospital of Sun Yat-sen University and The People’s Hospital of Chenzhou City between January 2012 and August 2020 were retrospectively investigated. The inclusion criteria were as follows: confirmed pathological diagnosis of pLELC, Eastern Cooperative Oncology Group performance status (ECOG PS) ≤ 2, and ≥2 regular treatment cycles. The exclusion criteria were as follows: had previous treatment after diagnosis, non first-line treatment, or lacked completed radiology or follow-up data. Finally, 68 patients were included in the study , PD-L1 expression , tumor location, clinical TNM (cTNM) stage, treatment mode, treatment cycle, treatment-related adverse events (TRAEs), and PFS. cTNM was determined according to the 8n Cancer . Based oThe chemotherapy group received platinum-based chemotherapy in combination with other non-platinum anticancer agents as doublet chemotherapy. Subsequently, non-platinum anti-cancer agents were administered as maintenance in some patients. In the first four treatment cycles, the chemoimmunotherapy group received a triple-drug regimen, including platinum, non-platinum anti-cancer agents, and PD-1 monoclonal antibody. Subsequently, the PD-1 monoclonal antibody was given as maintenance. All patients in the immunotherapy group received PD-1 monoclonal antibody monotherapy only. All treatment cycles were continuous and 3–4 weeks apart.Patients underwent a CT scan every 6–8 weeks to evaluate the treatment effect. The change in tumor burden was assessed using the Response Evaluation Criteria in Solid Tumors  . All radTRAEs were assessed using the National Cancer Institute Common Terminology Criteria for Adverse Events (version 5.0) .https://www.r-project.org/). Considering that the earliest date of immunotherapy patient data that could be collected was July, 2017, the primary endpoint was determined as the 2-year PFS in this study. PFS was defined as the time from the initiation of treatment to definite tumor progression or death. All follow-up data were collected until September 1, 2021. Patient age was transformed into a categorical variable (≥65 years old or <65 years old). Fisher’s exact test was used to determine significant differences between groups for categorical variables. PFS curves were completed using the Kaplan–Meier method and assessed using the log-rank test. The median and 95% confidence intervals (CIs) and p-values from log-rank tests are reported in figures. All P values were two-sided, and values <0.05 were considered statistically significant.All statistical analyses in this study were performed using R software v3.61 . However, there was no significant difference in 2-year PFS between the immunotherapy and chemotherapy groups  patients in the chemoimmunotherapy group and 5 (71.4%) in the immunotherapy group with PD-L1 expression ≥ 50% (There were no instances of grade 5 TRAEs in this study. Grade 3/4 TRAEs were reported in 14 (28.6%), 2 (28.6%), and 4 (33.3%) patients in the chemotherapy, immunotherapy, and chemoimmunotherapy groups, respectively Table 2.P=0.007), with an mPFS that was 5 months longer than chemotherapy.This is the first study to evaluate the efficacy and safety of first-line immunotherapy with or without chemotherapy in advanced pLELC. Our results showed that the 2-year PFS with chemoimmunotherapy was significantly better than that of chemotherapy  in the pembrolizumab group and 6.0 months  in the chemotherapy group. PFS was significantly longer in the pembrolizumab group than in the chemotherapy group  , 20. In The pattern of response observed in this study is consistent with the response to PD-1 inhibitors reported in other cancers . In the EGFR mutations and ALK rearrangement, are rarely detected in pLELC, indicating that the key carcinogens of LELC are not tobacco exposure or somatic cell driver mutations .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Serum concentrations of IL-10, leptin, calprotectin and CRP as well as in WBC count were in the same range in both studied groups. We observed direct statistically significant correlations between levels of IL-10 and CRP (p = 0.005), IL-10 and WBC (p = 0.045), TNF-α and WBC (p = 0.038), calprotectin and WBC (p < 0.001), chemerin and CRP (p < 0.001) as well as between NGAL and WBC (p = 0.002) in children with CMA. Conclusion: The use of a strict milk-free diet by children with CMA, resulting in the resolution of clinical symptoms of the disease, does not seem to extinguish the inflammation induced by the allergy. The findings of this study—elevated IL-6, TNF-α, resistin, chemerin and NGAL levels in patients with CMA—suggest that these parameters seem to be involved in the generation of a low-grade proinflammatory environment observed in cow‘s milk allergy and could be used to monitor the effectiveness of treatment.Background: The aim of the study was to establish whether the use of a strict milk-free diet in children with cow’s milk allergy, resulting in the resolution of clinical symptoms of the disease, also extinguishes the inflammatory reaction induced by the allergy. Methods: We examined 64 children (aged 3–6 years) with a diagnosed cow’s milk allergy who had been treated with an elimination diet for at least six months and showed remission of the disease’s clinical symptoms as a result of the treatment. The control group consisted of 30 healthy children of the same age following an unrestricted age-appropriate diet. Concentrations of cytokines, calprotectin, and adipokines  were determined in the serum samples obtained from the studied children by immunoenzymatic assays. Results: Patients with CMA had significantly higher median values of serum IL-6, TNF-α, resistin, chemerin and NGAL in comparison to the healthy children ( Cow’s milk allergy (CMA) is one of the most common food allergy forms in children, affecting about 2–5% of infants and young children ,2. CMA iDisturbances in the regulation of the immune response of the Th1/Th2 cells to antigen play a key role in the patophysiology of allergic diseases including food allergy. Lately, special attention has been paid to cytokines as the main regulators of cow’s milk allergy. Among them, IL-6, IL-10 and TNF-α play essential role. TNF-α with its pro-inflammatory properties can promote Th1 response, whereas IL-10 plays a role in Th1/Th2 cross-regulation. IL-10 produced by Th2 cells directly suppresses Th1 cells and can indirectly suppress Th2 cells activity ,9,10.The link between inflammation and adipose tissue has not yet been understood ,12. In rAdditionally, neutrophilic lipocalin associated with gelatinase (NGAL) which from a metabolic standpoint is also considered to be an adipokine (due to its high expression in adipose tissue) plays a possible role in neutrophilic inflammation ,29. BindCalprotectin, reported as an early signal mediating the cytokine cascade, is involved in an organism’s first response to inflammation. It acts through TLR-4 and activates nuclear factor-κB and other transcription factors, leading to increased production of metalloproteinases and proinflammatory cytokines ,35,36. PThus far, there has been limited research on serum pro-inflammatory cytokine and adipokine concentrations as well as calprotectin levels—essential modulators of inflammatory reaction and immunity—in children with cow’s milk allergy. The aim of the study was to investigate whether the use of a strict milk-free diet in children with cow‘s milk allergy, resulting in the resolution of clinical symptoms of the disease, also extinguishes the inflammatory reaction induced by the allergy. For that purpose, we examined serum concentrations of cytokines and adipokines related to inflammatory processes and their correlations with routinely determined parameters such as: white blood cell (WBC) count and C-reactive protein (CRP).This study was approved by the Ethics Committee of the Institute of Mother and Child (Prot. No. 19/2018) and was in accordance with the Helsinki Declaration of Principles. Written informed consent was obtained from all children’s parents. Children were recruited between May 2018 and July 2019 from a group of consecutive patients attending the Institute of Mother and Child in Warsaw (Poland). We examined prepubertal children with cow’s milk allergy treated with a dairy-free diet (n = 64) who remained under medical and dietary care. Cow’s milk protein allergy was diagnosed based on family history of allergy, clinical symptoms suggestive of allergy, measurement of serum cow’s milk-specific IgE concentrations (CM-specific IgE levels of less than 0.35 IU/mL were classified as undetectable), resolution of clinical symptoms after a 4-week diagnostic dairy-free diet. In children with IgE-mediated allergy, the therapeutic diet was continued. While in children with non-IgE-mediated CMA, allergy to cow’s milk was confirmed by re-introduction of dairy products to the diet which resulted in clinical symptoms.The inclusion criteria were: (a) diagnosed CMA, (b) following a milk-free diet for at least six months resulting in the disappearance of clinical symptoms of allergy, (c) being in the prepubertal period . Patients with acute and chronic infections were excluded. None of the children enrolled in the study were receiving an antibiotic/probiotic at the time of eligibility, time since the last infection treated both symptomatically or requiring antibiotic therapy was at least 3 months. The subjects with CMA had clinical symptoms from one system: gastrointestinal (12/64), skin (7/64), respiratory (30/64) or from many systems (15/64). Children were treated with casein or whey extensively hydrolyzed formulas (54%), soy-based formulas (26%) and plant-based milk substitutes (20%). We divided CMA patients into the IgE-dependent and IgE-independent subgroups resulting from the classification of immune reactions underlying food allergy. There were 41 (64%) IgE-mediated CMA and 23 (36%) non-IgE-mediated CMA patients. In our study, the levels of biochemical inflammatory markers were assessed in children with CMA who had remained on a strict elimination diet for at least 6 months, and 6 to 12 months had passed since the last food provocation attempt.The control group included prepubertal healthy children (n = 30) within the same age range as the CMA group—with a negative family history of allergy, no clinical symptoms suggestive of allergy, no abnormalities on physical examination, and following a traditional age-appropriate unrestricted diet. These were children who came to the gastroenterology out-patient clinic for evaluation of dietary balance.All studied children were followed up as outpatients, were normal weight children, and were not taking anti-inflammatory, anti-allergic or immune-modulating drugs/supplements (except for vitamin D supplementation) for at least three months to avoid their influence on the concentrations of biochemical markers of inflammation. All children with CMA were supplemented with vitamin D at a dose of 1000 (500–2000) IU/day depending on body weight and vitamin D supply in the diet. Healthy children (except for 5 cases) were also supplemented with vitamin D at a dose of 1000 (500–1500) IU/day.2). The obtained BMI value was related to age-and-gender specific BMI centile charts and BMI z-scores were calculated [Both allergic and healthy children had physical examinations performed, including height and weight measurements. Body mass index (BMI) was calculated as body weight (kg) divided by height squared , the CRP concentration was determined by standard laboratory methods using the biochemical Cobas Integra analyzer . The rest of the serum was frozen (−20 °C) in portions and stored (no longer than 2 months) for later examination of cytokines and adipokines using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions. All samples were tested in duplicate.For biochemical analyses, 3 mL of venous blood was taken in the morning hours. White blood cell count was determined as part of a routine test. In the serum obtained after centrifugation (1000× The concentration of IL-6 was assessed using High Sensitivity IL-6 (hs IL-6) Human ELISA kits from Abcam . The lowest detection limit was 0.81 pg/mL, intra-assay and inter-assay coefficient of variations (CV) were 4.4%, and 9.1%, respectively. Serum IL-10 level was detected using High Sensitivity IL-10 Human ELISA kits from Bender MedSystems GmbH . The minimal detection limit of this assay was 0.05 pg/mL, intra-assay and inter-assay CVs were 6.8% and 7.5%, respectively. The concentration of TNF-α was assessed using kits from DRG Instruments GmbH  with the limit of detection of 0.7 pg/mL, intra-assay precision between 4.6–6.6%, and inter-assay CV: 3.3–4.5%. The concentration of serum calprotectin was measured using kits from Calpro AS . This assay’s minimal detection limit was 5 ng/mL, intra-assay and inter-assay precision CVs were between 3.6-5.0% and 4.8 = 7.9%, respectively.Leptin concentration was determined using kits from DRG Instruments GmbH  with the lower detection limit of 0.7 ng/mL, intra-assay CV ranging between 4.2–7.3% and inter-assay CV between 3.7–9.1%. Circulating resistin was measured using kits from AdipoGen Life Science  with the limit of detection of 0.1 ng/mL, intra-assay CV between 2.9 and 5.2%, and inter-assay CV between 4.2–7.2%. Chemerin concentration was assessed using a kit from Mediagnost , the intra-assay and inter-assay CVs were 2.0–2.8% and 4.4–5.9%, respectively. The analytical sensitivity of this assay is 5 pg/mL. NGAL level was determined using a kit from BioPorto Diagnostics A/S , with within assay variability less than 10% and between assay variability less than 12%. This assay‘s minimal detection limit was 1.4 pg/mL.p value equal to or less than 0.05 was considered to be statistically significant.Statistical analyses were performed using IBM-SPSS software version 23.0 . The Kolmogorov–Smirnov test was used to test the normality of variable distribution. Baseline characteristics were presented as mean±SD for the variables with normal distribution or as median values and interquartile range (IQR) for non-normally distributed variables. Student‘s t-test for normally distributed variables and Mann–Whitney U test for non-parametric variables were applied to evaluate differences between the groups regarding anthropometric and biochemical parameters. Univariate correlation analysis was performed using the Spearman test. A All the children examined for the purpose of this study were Caucasian. There were 64 children (32.8% girls) aged 3 to 6 years with cow‘s milk allergy treated with a dairy-free diet for at least six months. Their results were compared with group of 30 healthy non-allergic children (33.3% girls) of the same age range, following a traditional unrestricted age-appropriate diet. Although the studied groups of children did not significantly differ in terms of age, the control children were slightly older than the children with CMA. Therefore, they had higher body weight and height. However, the BMI and BMI z-score indexes were comparable in both studied groups .p = 0.049), TNF-α (p < 0.001), resistin (p = 0.037), chemerin (p = 0.005), and NGAL (p < 0.001) in patients with CMA compared with the healthy children  (p < 0.001) and significantly higher eosinophils count in IgE mediated subgroup than in non-IgE mediated CMA patients (p < 0.001).There were no statistically significant differences in levels of biochemical parameters associated with inflammation between the subgroups of children with IgE-mediated CMA and with non-IgE-mediated CMA, except for the concentration of IL-6, which was higher in patients with IgE-mediated CMA (= 0.021) . Additiop < 0.05). Serum calprotectin was lower in patients with respiratory symptoms than in those with cutaneous or gastrointestinal symptoms by about 20–25% (p < 0.05). The values of IL-6 and TNF-α were comparable across the subgroups of patients with CMA. Regarding adipokines, we observed their elevated serum levels in patients with CMA who had clinical symptoms from many systems compared to those with clinical symptoms from one system. Due to the small sample size in the subgroups, these differences did not reach statistical significance, except for NGAL concentrations (p = 0.048).We performed an analysis of studied biomarkers in subgroups of patients with different clinical presentations . The higp = 0.125, p = 0.051, respectively). Due to the small number of patients in the subgroups, these differences did not reach statistical significance .We also analyzed serum concentrations of cytokines, calprotectin and adipokines in patients with different diet durations. We did not observe significant differences in these biomarkers between the three subgroups . Howeverp < 0.05) between WBC and IL-10, TNF-α and calprotectin levels as well as between the concentration of CRP and IL-10  and between NGAL and WBC count (p < 0.01)  .p < 0.01) and with CRP (p < 0.01), while chemerin level with WBC (p < 0.05) and with CRP (p < 0.01). However, leptin concentration was inversely correlated with WBC (p < 0.05). Moreover, we observed a significant positive correlation between serum levels of TNF-α and IL-10 , TNF-α, and chemerin  and between calprotectin and NGAL (r = 0.432 p = 0.001) in patients with CMA. In the group of healthy children we noticed significant relations between the concentration of chemerin and resistin  and between TNF-α and NGAL  (data not shown).In the control group, serum resistin was directly correlated with WBC .Considering that cow’s milk allergy is associated with persistent inflammation and the fact that, in some subjects, the symptoms of this disease may last many years, it seems that the search for new markers that can be useful in monitoring the dynamics of the inflammatory process is necessary. The possibility to use cytokines responsible for the allergic process related to CMA has been proposed in previous studies . In the α. In our studied patients with CMA, we observed above two-fold higher eosinophils count in the subgroups of IgE-mediated allergic patients than the non-IgE-mediated patients. There was no significant difference regarding eosinophils count between the groups of non IgE-mediated CMA patients and the healthy children. This is not surprising since eosinophilia is one of the markers that can, among others, indicate atopy.In patients with persistent CMA, the exposure to cow’s milk proteins could result in chronic allergic inflammation with its clinical consequences. Mast cells have essential immunoregulatory functions and play a significant role in immediate allergic and inflammatory reactions. Through their production of inflammatory mediators and cytokines, they contribute to the recruitment of eosinophils at the site of inflammation. Mediators released upon mast-cell degranulation, particularly histamine, could stimulate endothelial or epithelial cells to release a potent eosinophil chemoattractant, such as eotaxin. This causes the infiltration of eosinophils, along with basophils, into inflamed tissues. It is known that eosinophils as part of the immune system, when working correctly, could help fight disease and infection. Nevertheless, having too many activated eosinophils may contribute to disease pathology and the self-perpetuating cycle of inflammation. Such dysfunction leads to dysregulation of biological mechanisms involved with eosinophil recruitment and activation . The earThe effects of vitamin D on calcium-phosphate metabolism are relatively well understood. Since vitamin D receptors are present in cells of the immune system, e.g., neutrophils, macrophages, activated B and T lymphocytes and dendritic cells, attention is drawn in the immune response regulation. It has been proven that vitamin D decreases the concentration of proinflammatory cytokines , simultaneously increasing the concentration of anti-inflammatory cytokines . Thus, an adequate supply of this vitamin to children with inflammatory diseases, including cow’s milk protein allergy, appears be of importance. All children with CMA were supplemented with vitamin D and had a sufficient vitamin D status (serum 25-hydroxyvitamin D above 30 ng/mL). There were no differences in the levels of 25 OH-D in the subgroups of IgE mediated and non IgE mediated CMA patients.While analyzing the subgroups of patients with different clinical presentations, we observed elevated serum adipokine levels in patients with CMA who had clinical symptoms from many systems compared to those with clinical symptoms from one system. Additionally, we noticed the highest level of IL-10 in patients with gastrointestinal symptoms and decreased serum calprotectin in children with respiratory symptoms. As far as the duration of a milk-free diet is concerned, we observed a trend towards higher serum IL-6 and lower IL-10 concentrations with longer diet duration.p < 0.05) serum leptin compared to healthy children. The results from our study suggest that serum resistin increased but leptin seemed to be unaffected in children with CMA treated with a milk free diet.A dysregulated adipokine balance might also be an important link between inflammation and several disorders . Thus, wAnother adipokine, chemerin, was classified as both an adipokine and a chemoattractant for immune cells. Chemerin binding to a receptor (expressed on various immune cells) may regulate chemotaxis towards the site of inflammation. Recently, there have been intense studies on the axis between chemerin and its receptor (chemR23) and its multiple roles in the regulation of cell proliferation, migration and invasion ,43. In vElevated serum chemerin concentrations and a significant positive correlation of chemerin with the levels of inflammatory markers  were observed in several diseases ,46. ChemNeutrophil gelatinase-associated lipocalin is induced explicitly in damaged nephrons. Therefore, it was initially used for the early detection of acute kidney injury . RecentlSome studies mention NGAL as a useful parameter in distinguishing asthma-chronic obstructive (ACO) pulmonary disease from COPD ,54. PlasSeveral previous studies determined serum or fecal calprotectin levels in cases of several inflammatory diseases ,58,59. IAlthough the results of this study, indeed, do not yet create a direct clinical tool allowing for immediate practical application, in our opinion, they constitute solid ground for further investigations in this promising area. Considering that diagnostics of food allergy, especially IgE-independent food allergy, is an important issue, the results of our study contribute to the search for markers facilitating diagnostics and monitoring effectiveness of therapy of this type of allergy. Having in mind the potential practical importance of the possible outcomes of subsequent research, further investigations seem to be necessary and fully justified.This study has several limitations. The main limitation may be related to the cross-sectional nature of our study, small sample size, and short-term follow-up. However, the studied group is homogenous, all subjects were Caucasian children aged 3 to 6 years. All children were in the prepubertal period, which eliminated the puberty’s influence on the concentration of tested adipokines. Overall, the sample size of 64 is comparable to the scope of other published studies on CMA in children ,41,42. DIn conclusion, the use of a strict milk-free diet by children with CMA, resulting in the resolution of the disease’s clinical symptoms, does not seem to extinguish the inflammation induced by the allergy. The findings of elevated IL-6, TNF-α, resistin, chemerin and NGAL levels in patients with CMA, suggest that these cytokines seem to be involved in the generation of a low-grade proinflammatory environment observed in cow’s milk allergy. It is also possible that they could be used to monitor the effectiveness of the treatment. The obtained results will contribute to broadening the knowledge on the role of cytokines and adipokines and their interrelationships in the mechanisms of inflammation in children with allergies. However, further extensive investigations are required to explore the effect of cytokines on inflammation and its potential utility in clinical applications.


Conditions have been identified that allow for the use of both alkenyl-bromides and -triflates. In addition, the hydrogenation creates another opportunity for stereocontrol, thus allowing access to multiple stereoisomers of the product. Finally, the method is demonstrated in the streamlined synthesis of a biologically relevant molecule.Through the combination of a Ni-catalyzed alkene alkenylboration followed by hydrogenation, the synthesis of congested Csp 3–Csp3-bonds can be achieved.Through the combination of a Ni-catalyzed alkene alkenylboration followed by hydrogenation, the synthesis of congested Csp While much progress has been made in the development of alkyl–alkyl cross-coupling reactions, synthesis of congested Csp3–Csp3 bonds remains a formidable challenge  are converted to more complex structures with control of diastereoselectivity and regioselectivity in one step, and the reactivity of the resulting C–B bond, which can be easily transformed into C–O, C–N and C–C bonds thus allowing for diverse product formation.6 One of the key aspects of the Ni-catalyzed arylboration reaction is that sterically demanding di- and tri-substituted unactivated alkenes can be used. In these reactions, a stereodefined tertiary alkyl-Ni-complex is generated by borylnickelation of an alkene, which undergoes facile reaction with an arylbromide.5 To address the aforementioned challenge of making Csp3–Csp3 bonds, we envisioned a net alkylboration that would merge an alkenylboration and subsequent hydrogenation . Both sterically demanding trisubstituted and 1,1-disubstituted alkenes function well in the process to generate a congested C–C bond between a quaternary carbon and a β-branched primary carbon. A focus of these efforts was on the synthesis of saturated nitrogen containing heterocycles, which are of value in medicinal chemistry.13 Particularly notable are examples 12 and 13 as these are generated as single observable diastereomers. In addition, reaction to produce 6, occurred with high selectivity for addition on the face opposite the ester. Reactions of more strained alkenes generally result in higher yield (compare product 11 with 13/14). Finally, with 1-bromo-2-methylpropene the reaction of cyclic 1,2-disubstiuted alkenes were also investigated, which allowed for the synthesis of syn-1,2-substituted 5-membered ring carbo- and heterocycles.Under conditions optimized for arylboration of alkenes,15–19 the coupling proceeded to prepare the sterically congested bond of coupling between 2° and 3° fragments. In the case of products 23 and 26, the hydrogenation reaction led to formation of diastereomers with variable selectivity. The formal introduction of a 1° alkyl group can also be achieved with use of bromides 20a/b and 21. With the former example, both E and Z-alkenyl bromide work with equal efficiency. In addition, functional groups such as acetals (product 2) and silyl ethers were tolerated (product 28). Finally, the reaction can be performed on gram scale as demonstrated in the synthesis of 4.With respect to the alkenyl bromide component, the use of all substitution patterns worked. In the case of k1 is reduced due to steric hindrance, thus allowed k2 to predominate  was evaluated, a significant increase in yield relative to use of NiCl2(DME) was observed  were still observed  was formed as that observed in the reduction of the Bpin-derived substrate. Based on the stereochemistry of product 45, reduction likely occurs from the least hindered face, as shown in model 48. On the other hand, directed hydrogenation of the alcohol with Crabtree's catalyst led to formation of the opposite diastereomer (47), likely via intermediate 49.16 It is important to note that while the selectivities are modest, this strategy demonstrates that tuning of conditions can allow for stereodivergent synthesis. In addition, these examples demonstrate that three contiguous stereogenic centers can be prepared from simple components in a modular fashion.Hydrogenation with substrates that would result in diastereomers was probed more deeply . Hydroge51 was prepared though a brief sequence of five steps in 43% overall starting from 34 and 50. Thus, the demonstrated strategy offers an alternative to the established route that required eight steps.17Finally, the alkenylboration/hydrogenation sequence was used in the synthesis of drug like intermediates . Compoun3–Csp3 bonds by a formal alkylboration of unactivated alkenes is reported. The process was made possible by combining a Ni-catalyzed alkenylboration followed by hydrogenation. In addition, the hydrogenation could be tuned to achieve stereodivergent synthesis. Through the development of this process, we have demonstrated the utility of Ni-catalyzed carboboration for the generation of molecular complexity with high Csp3 content.In summary, the synthesis of sterically congested CspA. K. S., S. R. S and M. K. B. designed the study. A. K. S. and S. R. S. performed the experiements. A. K. S. and M. K. B. wrote the paper with input from S. R. S.There are no conflicts to declare.SC-012-D1SC00900A-s001SC-012-D1SC00900A-s002


D-dimer is a sensitive biomarker for cancer-associated thrombosis, but little is known about its significance on cancer therapeutics-related cardiac dysfunction (CTRCD).Consecutive 169 patients planned for cardiotoxic chemotherapy were enrolled and followed up for 12 months. All patients underwent echocardiography and blood test at baseline and at 3-, 6-, and 12 months.n = 37) and low D-dimer group (n = 132). Left ventricular ejection fraction (LVEF) decreased at 3- and 6 months after chemotherapy in high D-dimer group , but no change was observed in low D-dimer group. The occurrence of CTRCD within the 12-month follow-up period was higher in the high D-dimer group than in the low D-dimer group . Multivariable logistic regression analysis revealed that high D-dimer level at baseline was an independent predictor of the development of CTRCD .The patients were divided into two groups based on the level of D-dimer (>1.65 μg/ml or ≦ 1.65 μg/ml) at baseline before chemotherapy: high D-dimer group (We should pay more attention to elevated D-dimer levels not only as a sign of cancer-associated thrombosis but also the future occurrence of CTRCD. Recent advances in the diagnosis and treatment of cancers improve its prognosis. However, anticancer drugs, namely, anthracyclines, monoclonal antibodies, tyrosine kinase inhibitors, etc., induce cardiac dysfunction, resulting in poor prognosis in cancer survivors . SeveralD-dimer is a sensitive biomarker for cancer-associated thrombosis, but accumulating evidence suggests that pretreatment D-dimer can be used as a prognostic biomarker for patients with solid tumors . In cardAlthough D-dimer is a promising biomarker in the cardio-oncology field, little is known about the relationship between D-dimer and CTRCD. The present study aimed to evaluate the predictive impact of D-dimer before chemotherapy on the development of CTRCD.n = 33). The remaining 169 patients were divided into two groups based on the cut-off value of D-dimer, which was defined by the receiver operator characteristic curve analysis to detect the occurrence of CTRCD  inhibitors, tyrosine kinase inhibitors, and proteasome inhibitors, at Fukushima Medical University hospital from November 2016 to March 2019 . Patientof CTRCD .Hypertension was defined as a history of use of an antihypertensive drug or systolic blood pressure of ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg. Diabetes was defined as recent use of insulin treatment or hypoglycemic drug or hemoglobin A1c ≥6.5%. Dyslipidemia was defined as a history of use of cholesterol-lowering drugs, or triglyceride was ≥150 mg/dl, low-density lipoprotein cholesterol was ≥140 mg/dl, and/or high-density lipoprotein cholesterol was ≤ 40 mg/dl. A cumulative dose of anthracycline was expressed as a doxorubicin equivalent . Left ventricular (LV) EF was calculated using the modified Simpson's method according to the guideline from the American Society of Echocardiography and the European Association of Cardiovascular Imaging . The LV Cancer therapeutics-related cardiac dysfunction was defined as a decrease in EF by more than 10% points, to a value <53% . The LV High sensitivity cardiac troponin I (TnI) was measured using an assay based on Luminescent Oxygen Channeling Immunoassay technology and run on a Dimension EXL Integrated Chemistry System . B-type natriuretic peptide (BNP) levels were measured using a specific immunoradiometric assay . D-dimer was measured using a latex agglutination method .t-test was used for variables following a normal distribution, the Mann-Whitney U-test was used for variables of the non-normal distribution, and the chi-square test was used for categorical variables. The time course of EF  was evaluated using the Friedman test.All statistical analyses were performed using Prism 9  or R software packages version 3.6.3 . We used the Shapiro-Wilk test to discriminate which variables were normally or not normally distributed. Normally distributed variables were shown as mean ± SD. Non-normally distributed variables were indicated by a median with interquartile range. Category variables were shown in numbers and percentages. Student's p < 0.05 in the univariable analysis were entered into the multivariable analysis. A receiver operating characteristic curve analysis was performed to determine the optimal cut-off value of the D-dimer for predicting the occurrence of CTRCD. The p of 0.05 or less was defined as significant.Logistic regression analysis was performed to identify the variables to predict the occurrence of CTRCD. We selected variables relating to the general condition and cardiac function, i.e., age, echocardiographic parameters, use of anthracyclines, BNP, hemoglobin, estimated glomerular filtration ratio, and the elevation of D-dimer. The variables presenting p = 0.0005), a higher rate of ovarian/uterine cancer , and a higher rate of leukemia  than low D-dimer group. Echocardiographic data demonstrated that EF was slightly higher in the high D-dimer group . In laboratory data, the high D-dimer group showed lower hemoglobin values and higher BNP values.First, we performed a receiver operating characteristic curve analysis to identify the threshold level of D-dimer to predict the occurrence of CTRCD . A totalp = 0.03, p = 0.0015; 6 months: −4.8 ± 8.0 vs. −0.2 ± 6.2, p = 0.0004; 12 months: −4.5 ± 7.3 vs. −0.4 ± 6.6%, p = 0.0024).Time-dependent changes in EF are displayed in p = 0.0146). Multivariable logistic regression analysis revealed that LV end-diastolic volume index  and high D-dimer levels  before chemotherapy were independent predictors of the development of CTRCD .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We isolated and characterized tilapia-head chondroitin sulfate (TH-CS) and explored its biological activity and mechanisms of action as an oral supplement for nonalcoholic fatty liver disease (NAFLD) induced by a high-fat diet (HFD) in mice. The results showed that treatment with TH-CS for 8 weeks alleviated the development of NAFLD, as evidenced by the notable improvement in liver damage, blood lipid accumulation and insulin resistance (IR). Meanwhile, TH-CS treatment reduced the expression of proinflammatory cytokines and normalized oxidative stress. Additionally, the analysis of 16S rDNA sequencing revealed that TH-CS could restore gut microbiota balance and increase the relative abundance of short-chain fatty acid (SCFA)-producing bacteria. Furthermore, SCFAs produced by related bacteria can further improve lipid metabolism and IR by regulating lipid synthesis signals. In conclusion, TH-CS is an effective dietary supplement for the prevention of NAFLD, and may serve as a potential supplementary treatment for lipid-related metabolic syndrome. Nonalcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome characterized by fatty hyperaccumulation and steatosis of the liver without immoderate alcohol consumption. NAFLD is the commonest abnormal liver function disease at present, whose prevalence has doubled in the past 20 years, making it a health issue of global concern . FurtherAs a severe challenge in the field of modern medicine, the pathogenesis of NAFLD is complicated and sustained through the joint regulation of a range of environmental, nutritional, metabolic, and gut microbiota factors . The iniDiet control, nutrition optimization, exercise and drug treatment are the main conventional treatments of NAFLD. At present, the most widely used lipid-lowering drugs mainly include statins and 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, which can lower the cardiovascular disease incidence rates and mortality in high-risk groups. However, statins can adversely affect muscle due to the inhibition of ubiquitin formation . TherefoCS is a sulfated glycosaminoglycan that is mainly obtained from the cartilage, bone and cornea of animals, and is the product of substituting sulfate groups at different positions in chondroitin . CS has Tilapia is a freshwater fish mainly cultured in the world aquaculture industry, and its processed products are mainly frozen tilapia fillets. In tilapia processing, many byproducts, such as fish heads, skin and bone are usually discarded and unused, which has caused enormous environmental problems. However, tilapia heads are rich in CS and may be developed and utilized as raw materials for aquatic health food . Moreoveα) and free fatty acids (FFAs) were acquired from Shanghai Enzyme-linked Biotechnology Inc. . Commercial kits for aminotransferase (ALT), aminotransferase (AST), TC, TG, HDL-C, LDL-C, glutathione (GSH), superoxide dismutase (SOD) and malonaldehyde (MDA) were purchased from Nanjing Jiancheng Bioengineering Institute . Antibodies against SREBP-1c were obtained from Santa Cruz Biotechnology Inc. . Antibodies against AMP-activated protein kinase (AMPK), phospho-AMPK, peroxisome proliferator-activated receptor (PPARγ) and fatty acid synthase (FAS) were obtained from Cell Signaling Technology . Mouse anti-β-actin antibody and goat anti-mouse lgG-HRP secondary antibody were obtained from Abcam Inc. . All other chemicals and reagents used were of analytical grade.Tilapias were purchased from Hainan Xiangtai Fishery Co., LTD  and stored at −20 °C. High-fat feed was purchased from Hunan Slac Jingda Laboratory Animal Co., Ltd. . Enzyme-linked immunosorbent assay kits for insulin (INS), adiponectin (ADPN), leptin (LEP), IL-6, tumor necrosis factor-alpha (TNF-v/v) ethanol at the ratio of 1:10  for 1.5 h three times, with subsequent vacuum filtration. Next, the solid residues obtained from the filtration were added to 1% NaOH (w/v) at a ratio of 1:5. The mixture was incubated in a water bath at 50 °C for 7 h and intermittently stirred. After filtration, the filtrate was collected and the filter residue was treated again in the same way. The two filtrates were mixed and adjusted to pH 4 to remove acidic protein. Next, 0.1% (w/v) complex protease was added to the filtrate and kept at 50 °C for 3 h. The mixture was boiled for 10 min to inactivate the complex protease. Trichloroacetic acid was added to the mixture at a final concentration of 10% (w/v) to remove the protein. The precipitated protein was removed by centrifugation at 4000× g for 15 min at room temperature. The obtained supernatant was heated to 65 °C, and preheated H2O2 was added to react for 10 h, during which the pH was maintained at 5. Ethanol was added at a final concentration of 70% (v/v) to the mixture, incubated at 4 °C for 18 h to obtain the precipitate. The precipitate was collected by centrifugation at 6000× g for 10 min at room temperature. The collected precipitate was re-dissolved in distilled water to filter through a stirred ultrafiltration unit UFSC05001  to remove salt and other contaminants. The purified fractions were freeze-dried for further analysis. According to the method described by Kosakai et al. [The tilapia heads were homogenized after cleaning and sonicated with 95%  and then pressed to a transparent film. The film was scanned in the frequency range of 500–4000 cm−1 three times.The FT-IR spectrum of TH-CS was determined using an FT-IR spectrometer . The dried TH-CS freeze-dried powder was ground with potassium bromide powder at the ratio of 1:50  method by using a Shimadzu LC-10A instrument  equipped with a BRT105-104-102 (8 mm× 300 mm) column. A total of 0.05 M NaCl was selected as the mobile phase, which flowed at a 0.6 mL/min rate. Dextrans with different MWs  were used as standards to obtain calibration curves by plotting the logarithm of retention time against their respective MW. Eventually, the MW of TH-CS was calculated according to the calibration curve.2 flow and re-dissolved with 100 mL of ultrapure water. After the mixture was centrifuged at 10,000× g for 10 min, the obtained supernatant was analysed by an ICS5000 ion chromatograph  equipped with an electrochemical detector. The chromatographic conditions used in the experiment were as follows: column, DionexCarbopacTMPA20 (3 mm× 150 mm); mobile phase A: H2O; mobile phase B: 250 mM NaOH; mobile phase C: 50 mM NaOH and 500 mM sodium acetate; and flow rate, 0.3 mL/min. Standard monosaccharides, including mannose (Man), galactose , rhamnose (Rha), glucose (Glc), glucuronic acid (GlcA), arabinose (Ara), D-fructose (Fru), xylose (Xyl), fucose (Fuc) and glucosamine (GlcN) were analysed under the same conditions as a reference.The sample was treated following the method described by Corrado et al. with a few modifications to analyse monosaccharide composition . In brie2O. The spectrometer frequency for NMR was 600 MHz.NMR spectroscopy was conducted on a Bruker AVANCE 600 MHz spectrometer to characterize the chemical structure of TH-CS. Prior to the NMR analysis, 20 mg of sample was dissolved in 0.6 mL DMale C57BL/6 mice (18.0 ± 2.0 g) were obtained from TianQin Biotechnology Company of Changsha with Licensed ID: SCXK2019-0014 . The mice were kept in cages under conditions of 22 °C, 55% relative humidity, with a daily light/dark cycle of 12 h. Mice were adapted to these conditions for one week before the experiment.n = 8): the normal control group (“Control”), the HFD-induced NAFLD group (“Model”), the NAFLD simvastatin (ST) group (“ST”), and the NAFLD TH-CS group (“TH-CS”). The control group was fed a standard diet while the other three groups were fed a HFD. The composition of the standard diet was crude protein (18%), crude fat (4%), crude fiber (5%), coarse ash powder (8%), moisture content (10%), calcium (1.0–1.8%) and phosphorus (0.6–1.2%). HFD was composed of standard diet (78.8%), lard (10%), egg yolk powder (10%), cholesterol (1%) and sodium cholate (0.2%). The mice in the ST group were treated orally with ST at a dosage of 3 mg/kg/day, and those in the TH-CS group were treated orally with 80 mg/kg/day TH-CS. The experiment lasted for 8 weeks, and the weight and body length were recorded once a week.Mice were distributed into four groups randomly  was calculated using Formula (3) to determine IR.α and FFA in the liver homogenate supernatant. The concentrations of serum TG, TC, HDL-C, LDL-C, ADPN, and LEP and enzyme activities of ALT and AST were also determined using commercial kits.According to the instructions, commercially available kits were used to measure the expression of SOD, GSH, MDA, IL-6, TNF-H&E staining: the 4% paraformaldehyde was used for liver tissue fixation. The liver tissues were then implanted into paraffin wax blocks after being dehydrated by a series of alcohol concentrations. These blocks were divided into 5-micrometer sections rapidly and stained with H&E for histological analysis. For oil red O staining, the liver tissues were fixed in 4% paraformaldehyde and dehydrated in sucrose solution. Next, they were embedded in the composite material and sliced. After staining with oil red, they were viewed at 400× magnification through an Olympus microscope .α-diversity among groups. To compare β-diversity, nonmetric multidimensional scaling (NMDS) was performed using OTUs for each sample based on Bray–Curtis. The 16S rDNA of caecal content samples were submitted to the NCBI database (SRA accession: PRJNA787458).A 16S-rDNA gene analysis was used to identify the gut microbiota diversity and composition of each group, which were collected from caecal content samples. Briefly, microbial DNA was extracted and checked by 1% agarose gel electrophoresis to evaluate its concentration and integrity, and after 16S amplification of the V3–V4 hypervariable region. The PCR products were sequenced on an Illumina HiSeq2500 platform to analyze the structure of gut microbiota after being quantified and purified. The chao1 and ACE were used to assess the w/v) and centrifuged at 13,000× g for 20 min at 4 °C. The internal standard was set by the mixture of filtered supernatant and 2-ethy acid. Next, the filtrate was injected into an Agilent 8890b-5977b gas chromatography-mass spectrometer  equipped with a flame ionization detector and an HP-FFAP column  capillary column. The setting temperature of the injector and detector was 260 °C. The oven temperature was initially set at 80 °C, then raised to 120 °C at a rate of 40 °C/min, raised to 230 °C at a rate of 10 °C/min, and finally held at 230 °C for 3 min. The column flow rate of He which worked as the carrier gas was 1 mL/min and the split ratio was 10:1. A standard curve was constructed with different concentrations of a standard mix containing acetate, propionate, butyric acid, isobutyrate, pentanoic acid and isovaleric acid.Gas chromatography was used to quantitatively analyse the concentrations of SCFAs, which were collected from caecal content samples. The samples were homogenized in ultrapure water at a ratio of 1:10 . Next, single-stranded cDNA was generated from 1 μg total RNA by reverse transcription by using a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific). A SuperReal PreMix Plus (SYBR Green) (TIANGEN Biotech) was used for quantitative PCR amplification. PCR was performed in a 20-microliter system. The gene-specific primers used are shown in The concentration of isolated total protein of liver tissue homogenate was measured by BCA assay . Subsequently, tissue lysates with equal protein amounts were separated by SDS-polyacrylamide gel electrophoresis  and blotted to PVDF membranes, which were incubated for 12 h with different specific primary antibodies after blocking with 5% nonfat milk solution. The membranes were then incubated with a peroxidase-labeled secondary antibody, and an ultrasensitive ECL Chemiluminescence Kit  was used. Western blot bands were visualized using a Tanon-5200 multifunctional imaging system  and the relative density of the bands was quantified by optical density using Quantity One.p-value was set to 0.05 and 0.01 for significant or extremely significant differences.Data are expressed as the mean ± SEM. Analysis of variance (ANOVA) was used to identify the significant differences between groups. For all analyses, the critical After calculation, the TH-CS yield in the present study was 0.44 ± 0.08% and the purity was 78.32 ± 0.14% (data not shown). Numerous studies have shown that the MW of polysaccharides is closely related to their biological activity. Specifically, polysaccharides with high MW generally have very poor water solubility, which makes it difficult to enter the organism to exert pharmacological effects and hinder their bioactivity . The HPG−1 could be ascribed to the O-H stretching vibrations, while the peak at 2931.80 cm−1 was attributed to the C-H stretching vibrations of -CH2 groups in the polysaccharide [−1 may have been caused by the C=O stretching vibration in the ester carbonyl [−1 was attributed to the S=O asymmetric stretching vibration, revealing the presence of sulfates in the TH-CS [−1 indicated that the sulfate groups of TH-CS were bound to N-acetyl-galactosamine  in the C6 position, which illustrates the presence of chondroitin-6-sulfate (CS-C) in TH-CS [The FT-IR spectrum of the TH-CS is shown in ccharide . The strcarbonyl . The sighe TH-CS . The C-Oin TH-CS .As shown in 1H NMR signals are concentrated in the region from 1.5 to 5.0 ppm. The signal at 1.99 ppm indicated the methyl group of the GalNAc structure [NMR is a fast method that provides an overview of TH-CS composition E. Typicatructure . Furthertructure . HoweverIn this study, the body weight, food intake, liver weight and body length of the mice were measured. As depicted in At the end of the experiment, Lee’s index and liver index values were dramatically higher in the model group than in the control group C,D. By cThe levels of AST and ALT are widely regarded as the “gold standard” indication of liver function . IntraceIR is at the core of the pathogenesis of metabolic syndrome. Therefore, controlling IR is the first but most crucial step in the treatment of NAFLD . HOMA-IRWe evaluated whether the administration of TH-CS ameliorated the abnormal lipid metabolism in mice caused by HFD consumption. TG and TC are mainly synthesized in the liver, and the long-term intake of HFD results in TG and TC synthesis and secretion disorders. This imbalance of lipid metabolism leads to an increase in serum TG, TC and LDL-C levels and a decrease in HDL-C levels. As shown in α through the lysosomal pathway to promote hepatic lipotoxicity [FFAs seem to be influential mediators of lipotoxicity and potential cellular toxins, which can prompt excessive fat accumulation through IR. In addition, FFA stimulates the expression of TNF-toxicity . As reflAs shown in Liver histology was obtained by staining with H&E and oil red O. The mice in the model group developed pronounced steatosis and vacuolization when compared with control group in H&E sections B. The moκB signaling pathway, are activated by the influx of excess nutrients into the metabolic pathway and trigger the classic inflammatory pathway. The activation of the inflammatory pathway promotes the increase of proinflammatory cytokines, such as IL-6, IL-1, and TNF-α [α and IL-6 were significantly higher in mice in the model group than in mice in the control group (α and IL-6 levels.The deterioration of NAFLD is closely related to the development of inflammation. Downstream signals of Toll-like receptors, such as the NF-nd TNF-α . After 8ol group A,B. BothThe levels of hepatic SOD, MDA and GSH were measured to investigate the role of oxidative stress in NAFLD C–E. As tThe incidence of NAFLD is intimately associated with obesity, excessive lipid accumulation and IR. As an endocrine organ, adipose tissue secretes cytokines, such as LEP and ADPN, which are essential in the progression of NAFLD. The mean levels of serum ADPN and LEP for the four groups of mice are shown in α-diversity was measured with the Chao1 (β-diversity was used to analyse the overall structural changes in the gut microbiota. The results of the NMDS-based β-diversity analysis indicated that the overall structure of the gut microbiota of the NAFLD mice was significantly changed, while the TH-CS-administered group clustered separately from the model group and shifted toward the control group (As an essential factor in the development of and recovery from NAFLD, the gut microbiota recent area of significant research focus. Therefore, we explored the impact of TH-CS on the microbiota’s diversity and composition. To evaluate the alterations in the diversity of the bacterial species among the four groups, the he Chao1 A and ACEhe Chao1 B indicesol group C. These Bacteroidetes, Firmicutes and Proteobacteria accounted for the largest proportion. The relative abundance of the gut microbiota in the NAFLD mice was significantly different from that in the normal mice. The abundance of Bacteroides rose, while those of Firmicutes and Proteobacteria decreased to varying degrees in the ST and TH-CS groups when compared with the model group. Subsequently, we identified distinctions between the four groups at the genus level. By comparison with model group, the ratio of the genera Faecalibaculum, Pseudomonas and Romboutsia decreased substantially, while the relative abundance of SCFA-producing bacteria, such as Butyricimonas, Odoribacter and Prevotellaceae, was enhanced in the TH-CS group (In the meantime, further analysis was performed to reveal the detailed differences in the relative abundance of bacterial communities at the phylum and genus levels. CS group E. MoreovSCFAs are final products of the indigestible foods fermented by intestinal microbes and play a role in regulating balance, inflammatory processes and obesity. Therefore, the types and contents of SCFAs of the mice’s caecal contents were measured at the end of the experiment. The total SCFA content in the model group was reduced when compared with that of the normal mice, but it was significantly higher in the TH-CS group and ST group than in the model group A. SpecifTo investigate the underlying molecular mechanism of lipid metabolism modulated by TH-CS, the expression of SCFA receptors and hepatic lipid metabolism genes were detected in the liver via qPCR and WB methods C–E.GPR41 and GPR43 are a couple of G protein-coupled receptors activated by SCFAs that are related to lipid synthesis. The expression levels of GPR41 and GPR43 were increased significantly in the livers of the TH-CS-treated mice compared with the NAFLD mice.γ is closely associated with obesity, adipocyte differentiation and IR. Our research showed that the expression of PPARγ in the livers of the NAFLD mice was significantly increased, whereas the expression of PPARγ in the TH-CS group was noticeably decreased.Because the AMPK-SREBP1 pathway plays an essential role in lipid metabolism, we explored whether the alleviating effect of TH-CS on NAFLD was associated with the expression of AMPK, the phosphorylation of AMPK, SREBP-1c and its downstream target genes FAS in the HFD-fed mice. AMPK is not only a cellular sensor that is indispensable to the restoration of cellular energy homeostasis; it is also a central regulator of multiple metabolic pathways. Therefore, it is considered as a therapeutic target for NAFLD . The expWith the prevalence of sedentary lifestyles and HFD, the incidence of NAFLD increases, making it a significant health concern. Thus, it is urgent to develop specific therapeutic drugs for NAFLD to reverse the occurrence and development of NAFLD. In recent years, various polysaccharides have been proven to be useful in fatty liver, hyperlipidemia and hyperglycemia . CS is rThe pathogenesis of NAFLD is generally considered to include a two-hit model. Abnormal hepatocellular lipid accumulation and IR are the first “hit” . With anDue to the causal relationship between liver fat accumulation and IR, IR has become a key factor in the first “hit” of NAFLD. When IR occurs, the insulin regulation of fat metabolism is weakened, resulting in an increase in FFAs released by adipose tissue decomposition . Increasβ and TNF-α in NAFLD rats [α and IL-6 in the NAFLD mice, suggesting that the TH-CS effectively protected liver cells from the inflammatory effects caused by HFD intake. Moreover, according to one report, CS derived from skate cartilage ameliorated hyperlipidaemia-induced oxidative stress [The “second hit” of NAFLD is considered to be linked to inflammation and oxidative stress. Based on the accumulation of TGs, inflammation and oxidative stress reactions occur in the liver, leading to hepatocyte degeneration and necrosis. According to a previous study, the intake of sulfated polysaccharides derived from brown seaweeds decreased the hepatic mRNA expression of IL-1FLD rats . In agree stress . Similarα- and β-diversity analyses, it could be seen that TH-CS significantly reshaped the gut microbiota structure and enhanced the microbial diversity of the NAFLD mice. In addition, the TH-CS significantly modified the composition of the gut microbiota at different levels of classification. In particular, TH-CS supplementation could significantly enhance the relative abundance of SCFA-producing bacteria, such as Butyricimonas, Odoribacter and Prevotellaceae. It was previously reported that polysaccharides cannot be degraded in the gastrointestinal tract after oral administration, but that they can be fermented by the gut microbiota and generate SCFAs to regulate inflammation and lipid metabolism, which is a vital mechanism for indigestible polysaccharides in the prevention of HFD-induced NAFLD [Many studies have demonstrated that the development of NAFLD is directly related to imbalances in the gut microbiota. Wu et al. prepared abalone sulfated polysaccharide and its desulfurization products and analyzed the contribution of sulfate groups through a mouse model. They found that the presence of sulfate groups could lead to some differences in the gut microbiota and its metabolites, thus forming differences in biological activity with other polysaccharides . In the ed NAFLD . In the γ is another key transcription factor; it is a nuclear receptor that can regulate lipid metabolism disorder by modulating free fatty acids. At the same time, PPARγ is related to the regulation of obesity factors, such as ADPN and LEP; changes in obesity factors are the best predictors of insulin sensitivity [γ in their liver tissue. The above results suggest that TH-CS can adjust the secretion of FFAs and obesity factors by decreasing the mRNA and protein levels of PPARγ, thereby improving IR.It is widely known that the gut microbiota and its metabolites, such as SCFAs, play a vital role in regulating lipid metabolism. In particular, SCFAs can offer energy to the host and regulate the expression of lipid-metabolism-related proteins as signal molecules . Severalsitivity . In the In this study, we extracted TH-CS from tilapia heads and explored its lipid-lowering effects and mechanisms in a HFD-induced NAFLD mouse model. The TH-CS exerted a protective effect against IR and lipid metabolism disorder. At the same time, the TH-CS treatment significantly reduced inflammatory responses in the liver and oxidative stress. These effects appear to have been mediated by the regulation of the gut–liver axis homeostasis modulation in the NAFLD mice, i.e., the gut microbiota, microbial metabolites and related lipid metabolism signaling pathways. TH-CS could modulate the structure and composition of the gut microbiota as well as stimulating related bacteria to produce SCFAs. Moreover, after degradation to SCFAs by the gut microbiota, the TH-CS regulated the AMPK-SREBP1 signaling pathway and the level of PPARγ in the liver to alleviate lipid accumulation and IR in the NAFLD mice. In short, we demonstrated that TH-CS can effectively alleviate HFD-induced NAFLD in mice. Our findings support the comprehensive utilization of the byproducts of aquatic animal processing and provide a theoretical basis for the use of TH-CS as a nutritional supplement for ameliorating NAFLD.